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Abstract

The prediction of allergen cross-reactivity is currently largely based on linear sequence
data, but will soon include 3D information on homology among surface exposed residues.
To evaluate procedures for these predictions, we need ways to quantitatively assess
actual cross-reactivity between two allergens. Three parameters are mentioned: 1)
the fraction of the epitopes that is cross-reactive; 2) the fraction of IgE that is
cross-reactive; 3) the relative affinity of the interaction between IgE and the two
allergens. This editorial briefly compares direct binding protocols with the often
more appropriate reciprocal inhibition protocols. The latter type of protocol provides
information on symmetric versus asymmetric cross-reactivity, and thus on the distinction
between complete (= sensitising) allergens versus incomplete, cross-reacting allergens.
The need to define the affinity threshold of the assay and a caveat on the use of
serum pools are also discussed.

Commentary

In a paper recently published in this Journal, the question was raised whether a fungus
considered for biological pest control (Beauvaria bassiana) could elicit allergic
reactions due to cross-reactive IgE antibodies induced by allergens from known allergenic
fungi [1]. Based on homology in the amino acid sequence, four potentially cross-reactive proteins
were cloned, expressed in E coli and tested for IgE (cross)reactivity using sera from
patients with known fungal allergies. Support for (cross)reactivity was found for
two of these four proteins. The two (cross)reactive proteins had the highest sequence
homology to known allergens: the enolase was 85% sequence-identical to the Alternaria
enolase known as Alt a 6 and the aldehyde dehydrogenase was 71% sequence-identical
to the Alternaria dehydrogenase Alt a 10. The two proteins with no demonstrable (cross)reactivity
had sequence identities of 51 and 60% to two Aspergillus fumigatus proteins. It is
tempting to conclude that this result supports the notion that sequence identity is
a useful predictor of cross-reactivity.

A few comments on the prediction on cross-reactivity as illustrated by this study.
As the authors point out, the number of sera used to test for cross-reactivity was
small (N = 20, tested as 10 pools of 2; a caveat on the use of serum pools will be
discussed later). Moreover, the clinical history of the patients is not specified,
which is particularly relevant in view of the quite distinct modes of allergen exposure
in case of invasive aspergillosis as compared to airborne Alternaria. IgE reactivity
was investigated by immunoblotting of crude E. coli extracts as the source of the
recombinant proteins, which is useful but not ideal since it is known to be inefficient
for some allergens. Lastly, as discussed in more detail below, cross-reactive potential,
particularly in case of polyclonal antibodies, is more reliably assessed by inhibition
tests than by direct binding tests.

Cross-reactivity and allergenicity

For various reasons, often related to regulatory safety issues, a discussion is ongoing
on prediction of allergenicity. This involves both prediction of de novo allergenicity
as well as prediction of cross-reactivity. The latter, prediction of allergen cross-reactivity,
is the topic of this communication, with emphasis on quantitative and methodological
aspects.

Clinically, allergic cross-reactivity is often encountered as symptoms without prior
exposure. Another common clinical situation is the occurrence of symptoms upon exposure
to allergenic sources that are unlikely to sensitise, such as apples. In Northern
Europe it is rare to find apple allergy in the absence of birch allergy. The major
birch pollen allergen acts as the sensitizer or primary allergen, which by definition
is able to trigger the immune system to produce IgE antibodies. The homologous protein
in apple Mal d 1 is an incomplete allergen, because it is unable (or: extremely inefficient)
to induce IgE antibodies, but is able to elicit symptoms due to its ability to trigger
mast cells loaded with IgE anti-Bet v 1.

Cross-reactivity is sometimes seen as a property of a subgroup of antibodies: antibodies
to some epitopes (recurring epitopes such as cross-reactive carbohydrate determinants
(CCDs [2]) are more likely to be cross-reactive than antibodies to other epitopes. However,
it is often more appropriate to use cross-reactivity to describe a relation between
two allergens (which I will refer to as Ag1 and Ag2; alternatively, I will use the
birch allergen Bet v 1 and the cross-reactive apple allergen Mal d 1 as examples):
the closer the similarity between two allergens, the more likely it is to find a cross-reactive
antibody. In either case, the concept of cross-reactivity concerns (at least) three
rather than two reagents: two allergens and an antibody. Since it is impossible to
test all antibodies, we have to live with the frustrating thought that it is impossible
to prove that two allergens completely lack cross-reactivity. Conversely, it is also
impossible to prove that they are fully cross-reactive. It is all a matter of probability,
which in many cases is either very close to 0 or very close to 1. In many other cases
it is, however, easy to demonstrate some cross-reactivity.

Prediction of cross-reactivity from the amino acid sequence

Antibodies largely bind to surface patches of folded proteins (epitopes), so knowledge
of the full 3D structures of the target protein and the related allergens would clearly
be an advantage for such a prediction and now more commonly available (or the 3D structures
can be reliably predicted). Undoubtedly, cross-reactivity prediction algorithms will
be developed in which such information is incorporated. However, sufficiently reliable
information on the relevant 3D structures is often not available and the prediction
has to be based on the linear amino acid sequence. One of the points of debate is
whether short stretches of 6–8 fully identical amino acids are reliable predictors
and should be used in combination with partial amino acid identity of longer stretches
(typically more than 35% identity between stretches of 80 amino acids [3]).

Other issues related to prediction of cross-reactivity, such as the repertoire-modifying
effect of a human homologue, the contribution of post-translational modification (particularly
non-mammalian glycosylation patterns), the possibilities and limitations of peptides
as epitope mimics and the intriguing question whether IgE antibodies tend to be more
cross-reactive than IgG antibodies, with its possible link with positive and negative
regulation of B cells by IgE versus IgG antibodies, have been discussed elsewhere
[4-6].

Symmetric versus asymmetric cross-reactivity

Some situations of allergen cross-reactivity are almost trivial, such as the cross-reactivity
between major allergens of botanically-related grasses [7] and between major dust mite allergens. Without information on allergen exposure it
is then virtually impossible to decide which allergen is the sensitizer. Symmetric
cross-reactivity a likely possibility: both allergens in the couple can sensitize
and both can largely (but not completely) inhibit the binding of IgE to the other
allergen (figure 1A). In the birch/apple situation the situation is different (at least in Northern Europe)
[8]. Cross-reactivity is asymmetric (figure 1B), as can be demonstrated in vitro by reciprocal IgE antibody neutralization. The
usual finding is that birch allergen inhibits IgE binding to the apple allergen similar
to or even better than the inhibition found by using equimolar amounts of the apple
allergen as inhibitor, whereas the apple allergen only partially inhibits IgE binding
to the birch allergen.

Issues on the quantification of the degree of cross-reactivity of two allergens

First, a semantic issue: polyclonal versus monoclonal cross-reactivity. The word "cross-reactivity"
is used differently in the polyclonal situation and the monoclonal situation. Traditionally,
it is used to describe the

polyclonal

situation as encountered in the body. Many different antibodies recognize Ag1 (for
example: Bet v 1). Some of these antibodies react with Ag2 (Mal d 1). The degree of
cross-reactivity can be expressed as the fraction of anti-Bet v 1 antibodies that
react with Mal d 1. This fraction will vary between individuals and usually varies
within one individual in time. A cross-reactive polyclonal antiserum can often be
made mono-specific by absorption with either Ag1 or Ag2. When this type of cross-reactivity
assessment is applied to a single

monoclonal

antibody, the unavoidable outcome would seem to be either: "fully cross-reactive"
or: "not cross-reactive". This is intuitively unsatisfactory, since "fully cross-reactive"
suggests equal reactivity of the antibody with Ag1 and Ag2, whereas in most cases
the antibody will have different affinities for, and thus preferentially react with,
either Ag1 or Ag2. An important qualitative modifier (which is mostly not specified
in papers on cross-reactivity) is the affinity threshold, which depends on the read-out
system and the concentrations used for testing. The effects of affinity are most visible
in the monoclonal situation, but also play a role in the polyclonal situation. In
general, a cross-reactive antigen will have a lower affinity than the antigen that
induced the antibody response. It is not clear below which affinity the cross-reactivity
becomes irrelevant, but it is important to appreciate that there is a grey area. In
clinical terms, a low affinity may translate into a high threshold for the cross-reactive
allergen and/or milder symptoms.

In biological systems (e.g. skin test, cellular in vitro assays), assessment of cross-reactivity
largely depends on direct testing, and thus on statistical associations. This is particularly
unconvincing if not only the antibodies are polyclonal, but also the allergens are
tested as allergen extracts (i.e. allergen mixtures). More reliable analysis is possible
in vitro. In the case of monoclonal antibodies, direct binding assays can be used
(figure 2). Also with polyclonal antibodies (typically from human serum) direct binding assays
can be used and could, theoretically, provide information on one of the parameters
in cross-reactivity assessment: the fraction of the epitopes that is cross-reactive.
This is reflected in the relative IgE binding obtained at saturating IgE antibody
doses (which corresponds to the vertical distance between the high-dose segments of
two dose-response curves as indicated in figure 2). In practice, this is not a trivial analysis. Moreover, such a direct-binding test
does not discriminate between cross-reacting and non-crossreacting IgE. Cross-reactivity
can be proven (and to a certain extent quantified) by reciprocal inhibition systems,
preferably with at least one purified single allergen (figure 3). The two other parameters in cross-reactivity assessment can be derived from such
measurements: 1) the fraction of IgE that is cross-reactive (the vertical distance
between the homologous and heterologous dose response curves when testing the complete
allergen on the solid phase at saturating inhibitor levels as indicated in figure
3A) and 2) the relative affinity of the interaction between IgE and the two allergens (the
horizontal distance between the homologous and heterologous dose response curves when
testing the incomplete allergen on the solid phase, as indicated in figure 3B).

Figure 2. If a serum with cross-reactive antibody to the birch allergen Bet v 1 is incubated
with Bet v 1 on the solid-phase, the binding curve is different in two ways: 1) it
rises to a higher level and 2) it is shifted to the left compared to the binding observed
with the cross-reactive apple allergen Mal d 1 on the solid phase. The first observation
reflects that only a fraction of the epitopes is cross-reactive (in this example:
50%, as indicated by the vertical arrow). The second observation reflects that only
a fraction of the antibodies is cross-reactive, but also that the affinity is usually
lower for the cross-reactive allergen (Mal d 1) compared to the sensitising allergen
(Bet v 1). In these model calculations, the concentration of Bet v 1 epitopes is set
at 1; 50% of these epitopes are assumed have a cross-reactive homologue in Mal d 1;
40% of the IgE antibodies are assumed to be cross-reactive. The upper curve represents
binding to Bet v 1, assuming a dissociation constant KD equal to 1. The next 3 curves represent binding to Mal d 1 at decreasing affinities
(KD equal to 1, 5 and 25, respectively. Note that this type of experiment does not prove
cross-reactivity. The observed binding could in theory also be due to co-sensitization.
This can be investigated by inhibition assays (see figure 3).

Figure 3. Model calculations illustrating the use of cross-inhibition to demonstrate cross-reactivity
and to distinguish the primary sensitising birch allergen Bet v 1 from the cross-reactive
(incomplete, non-sensitizing) apple allergen Mal d 1. The type of experiment shown
in figure 3A indicates what percentage of the IgE antibodies is cross-reactive (in
this example: 40%, as indicated by the vertical arrow). The type of experiment shown
in figure 3B shows that Bet v 1 inhibits all antibodies to Mal d 1 and provides a
crude estimate of the relative affinity of the interaction of the IgE antibodies with
the two allergens (if the concentration of the antibody as well as the solid-phase
allergen is equal to or below the KD of the IgE-allergen interaction), as indicated by the two horizontal arrows. The parameter
setting correspond to those used in figure 2, using an IgE concentration of 1, which
corresponds to values for uninhibited binding of IgE of 0.2660 for IgE binding to
Bet v 1 in figure 3A (KD = 1), and for IgE binding to Mal d 1 of 0.0077, 0.0341 and 0.1118 (KD = 25, 5 or 1, respectively) in figure 3B.

A caveat on the use of serum pools

As discussed above, cross-reactivity is largely a probability feature. For this reason,
it is rarely meaningful to test only one single serum. It is preferable to test a
large number of individual sera and pool the results by conventional statistical procedures.
However, the use of a serum pool rather than a large number of individual sera is,
obviously, economical: fewer tests need to be performed and smaller volumes of (often
precious) allergic serum samples are needed. From the statistical point of view it
is important to inversely adjust the volumes of serum to be pooled according to their
antibody titre: the higher the titre, the smaller to contributing volume should be.
Optimally, the

amount

of antibody contributed by each serum donor should be similar (typically within a
factor 2). Without such a precaution, it is likely that the pool will behave like
a dilution of the strongest serum.